The proper evaluation of pathological specimens is of high importance to public health. An example is the detection and diagnosis of cancer. Often this is performed by a screening mechanism in which a cell specimen is obtained from a patient and detected under a microscope for abnormalities. While screening tests have decreased mortalities associated with cancer by preventing the development of invasive disease, it is nonetheless dependent on the ability of a technician viewing a specimen under a microscope to detect abnormal cells and structures in thick specimens or thick clusters of cells. Success depends in part on the images presented for evaluation. When the image is viewed through a conventional high-powered light microscope, the thickness and complexity of the specimen, the nature and concentration of the stain, the microscope optics objective and the illumination source all influence what is seen.
The conventional evaluation of pathological specimens using standard compound high-powered microscopes has a number of shortcomings. For example, the standard compound microscope using axial illumination is particularly ill-suited for clusters of cells. Thick or overlapping images can be distorted because of diffraction of the light and because of absorption by thick sections. Moreover, depth of focus is greatly reduced at high power, and it is difficult to visualize structures above or beyond a thin focal plane. Evaluation is made difficult by deficiencies in resolution, contrast, light penetration, and sharpness of image. As a consequence of the difficult and subjective nature of the test, there is a continuing problem with false negative readings and ambiguous readings in various cytopathology specimens. An example includes the Pap smear where a diagnosis is known as “atypical squamous cells of undetermined significance” (ASCUS).
U.S. patent application Ser. No. 09/104,133 (hereinafter the '133 application), discloses that oblique or multiple oblique illumination (MOI) of a specimen (or the oblique illumination equivalent achieved by tilting the specimen), especially a cytopathology specimen such as cervical Pap smear, is superior to axial illumination for purposes of ascertaining cellular detail of diagnostic significance. Oblique or MOI illumination can be performed using the microscope described in U.S. Pat. No. 5,345,333 (Greenberg). As described in the '133 application, MOI provides dramatic capabilities for penetrating thick cell clusters of cytopathology specimens and assessing individual cellular detail within clusters. Microscopic observation of thick cell clusters proved difficult as is well known to those skilled in the art using conventional axial illumination, as interpretation within the flat field is suboptimal. MOI provides a number of advantages, including increased intensity of light penetration through a wide range of focal planes within the clusters, higher resolutions, higher sharpness of the image, and the stereo view surprisingly and unexpectedly allowed for three-dimensional information to be realized. This proves invaluable for assessing cells and cellular detail in thick specimens or thick cell clusters.
It would be highly advantageous if, rather than depend solely on a human observer, the results of MOI-mediated microscopic examination could be processed by a computer to provide 3-dimensional images amenable to further computerized analysis for cellular detail. To achieve this goal, however, a number of significant challenges must be met. One challenge results from the nature of the image the computer “sees” and the relationship of that seen image to the actual image in the specimen under the microscope. The image seen by the computer is the sum of minute square (or dot) individual image components, pixels, regularly spaced over a flat surface. Each pixel stores, in computer-readable form, the frequency and intensity of light that impacted that pixel. Because, however, microscopes cannot be perfectly focused, the pixels seen by the computer will have received light not only from the focal plane of interest (i.e., a “virtual” thin slice of the specimen), but also specimen components just below and just above that plane. The present invention provides a method for “cleaning up” such images so that they more closely represent only the image in the focal plane of interest. To accomplish this goal, the computerized process uses a novel algorithm and analyzes a plurality of images of a single specimen plane, each image taken using a different source of oblique illumination. The resulting images can be stacked by the computer to provide a 3-dimensional image of the specimen, which image can either be displayed on a computer monitor or be further analyzed by a computer to quantitatively characterize components of cells in the specimen.